Polypeptides containing polymorphisms of the repeated regions of pertactin in Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, their use in diagnostics, and in immunogenic compositions

ABSTRACT

Pertactin (PRN) is an outer membrane protein expressed by  Bordetella pertussis, Bordetella parapertussis , and  Bordetella bronchiseptica , which induces protective immunity to  Bordetella  infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed the pertactin of  B. parapertussis  is invariant, whereas the pertactin of  B. pertussis  varies mostly in region I and  B. bronchiseptica  varies in both the repeated regions I and II. Compositions containing pertactins and pertactin fragments containing variant sequences in these regions are useful as immunogenic compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims the benefit of U.S. ProvisionalApplication Ser. No. 60/206,969, filed May 25, 2000 (attorney docket no.03495.6047) The entire disclosure of this application is relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to proteins and polypeptides of the Bordetellaouter membrane protein called pertactin and the polynucleotides thatencode them. This invention also relates to the use of these proteinsand polypeptides in immunogenic compositions, diagnostic methods, anddiagnostic kits.

The genus Bordetella includes seven species. The most studied speciesare B. pertussis, B. parapertussis, and B. bronchiseptica. B. pertussisis responsible for respiratory infections only in humans. B.parapertussis causes infections in humans and sheep, and B.bronchiseptica infects many animal species, including humans.

These pathogens produce an array of virulence factors, the synthesis ofwhich is regulated by the two-component, bvg AS (2, 21) system. Thesefactors include toxins, such as pertussis toxin, which is the only toxinspecific to B. pertussis, tracheal cytotoxin, adenylatecyclase-hemolysin, and adhesins, such as filamentous hemagglutinin,fimbriae, and pertactin (PRN).

PRN is an outer membrane protein with an apparent molecular weight of 69kDa in B. pertussis, 70 kDa in B. parapertussis, and 68 kDa in B.bronchiseptica (5, 14, 15). The precursors of PRN are 91.5 kDa, 93 kDa,and 92.5 kDa in size, respectively. In B. pertussis, PRN has beendemonstrated to be an agglutinogen (4), promoting attachment to certaineukaryotic cells via an Arg-Gly-Asp (RGD) motif (13).

Antibodies specific for the B. bronchiseptica PRN are detected at hightiter in immunized piglets, whereas few if any of these antibodies aredetected in unprotected animals (19). Synthesis of the PRN by B.bronchiseptica correlates with protection (16). The immunization of miceor piglets with preparations of the PRN induces protective immunityagainst B. bronchiseptica infection (12, 19) and passively administeredmonoclonal antibodies prevent the death of animals challenged with B.bronchiseptica (16). B. pertussis PRN has also been shown to induceprotective immunity to intracerebral, aerosol and intranasal challengeswith B. pertussis in mice (11, 18, 20).

PRN is, therefore, now included in some acellular pertussis vaccines(i.e. vaccines composed of purified bacterial proteins) (9). However,the PRN proteins of these three species, although clearly related, havedifferent immunogenic properties. For example, preparations of B.pertussis PRN protect mice against intranasal B. pertussis challenge butnot against intranasal B. parapertussis challenge (11). They alsoprotect mice against intracerebral B. pertussis challenge, whereas theB. bronchiseptica PRN protein does not (18).

Comparison of the deduced amino acids of the three proteins, B.pertussis-PRN, B. parapertussis-PRN, and B. bronchiseptica-PRN, revealsa high degree of similarity, with the B. bronchiseptica and B.parapertussis proteins being more similar to each other than to the B.pertussis PRN protein (5, 14, 15).

The sequences of the three proteins differ in the number of repeats inregions I and II (FIG. 1 a). Using monoclonal antibodies, Charles etal., identified and characterized a protective immunodominant epitope ofthe P.69-PRN protein (6). This epitope spans the (Pro-Gln-Pro)5 repeatsequences located in region II. Differences in this region may accountfor the observation that sera from piglets that recognize B.bronchiseptica PRN do not react with B. pertussis PRN despite the highdegree of similarity between these proteins (12) and for the lack ofcross protection provided by the three proteins (11, 18, 20).

It has recently been shown that the PRN produced by clinical isolates ofB. pertussis varies. Sequences of the prn gene of various clinicalisolates revealed three major types of PRN variant (17). It has beensuggested that epidemics in the Netherlands result from changes in thesequences of the genes encoding PRN and PT because the proteins presentin the clinical isolates currently in circulation differ in sequencefrom those observed by the vaccinal strains used in this country (17).

For PRN of B. pertussis, all the observed amino acid differences arelocated in region I. The allelic prn types A=1 and C=3 are very similar,differing by only two amino acids, whereas type B=2 is quite different,having a five-amino acid insertion in the same region (17).

Only one type was found to differ in region II. This type (A*=6) isproduced by the B. pertussis WHO reference strain 18323 and one Frenchclinical isolate (3). It does not, however, seem to be common because ithas been detected in only one clinical isolate (3). The production bythis B. pertussis strain of this unusual type of PRN reflects the manycommon properties shared with the B. parapertussis and B. bronchisepticaspecies. No differences were found in the phenotype and behavior in theanimal model of B. pertussis clinical isolates with different PRN (3).

There is a need in the art for compositions containing proteins andpolypeptides of Bordetella pertactins that can be used in immunogeniccompositions to protect against Bordetella infection and to treatsubjects infected with Bordetella. Ideally, the proteins, polypeptides,and the polynucleotides that encode them would also be useful indiagnosing Bordetella infection and in kits for the diagnosis of suchinfection.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. In oneembodiment, this invention provides an immunogenic compositioncomprising a mixture of pertactins of Bordetella species, wherein saidcomposition comprises: (a) pertactin of Bordetella parapertussis, and(b) pertactin of Bordetella bronchiseptica, in amounts sufficient toinduce a humoral or cellular immune response against Bordetellaparapertussis and Bordetella bronchiseptica in an animal to which theimmunogenic composition is administered. The immunogenic composition canalso comprise pertactin of Bordetella pertussis in an amount sufficientto induce a humoral or cellular immune response against Bordetellapertussis in an animal to which the immunogenic composition isadministered.

In another embodiment, the immunogenic composition of the inventioncomprises a mixture of pertactins of Bordetella species or fragmentsthereof. Specifically, the mixture comprises a mixture of Bordetellabronchiseptica pertactin variants wherein each Bordetella bronchisepticapertactin variant comprises 6, 7, 8, or 9 repeating PQP amino acidsequences in Region II thereof. The Bordetella bronchiseptica pertactinvariants are present in amounts sufficient to induce a humoral orcellular immune response against Bordetella bronchiseptica in an animalto which the immunogenic composition is administered. This immunogeniccomposition can also comprise pertactins of Bordetella parapertussis,Bordetella pertussis, or mixtures thereof, in amounts sufficient toinduce a humoral or cellular immune response against Bordetellaparapertussis or Bordetella pertussis in an animal to which theimmunogenic composition is administered.

In a further embodiment of the invention, the immunogenic compositioncomprises a mixture of pertactins of Bordetella species or fragmentsthereof, wherein mixture comprises a mixture of Bordetellabronchiseptica pertactin variants, wherein each Bordetellabronchiseptica pertactin variant comprises 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof. The Bordetella bronchisepticapertactin variants are present in amounts sufficient to induce a humoralor cellular immune response against Bordetella bronchiseptica in ananimal to which the immunogenic composition is administered. Thisimmunogenic composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof, in amountssufficient to induce a humoral or cellular immune response againstBordetella parapertussis or Bordetella pertussis in an animal to whichthe immunogenic composition is administered.

The compositions of the invention can comprise a mixture of fragments ofthe pertactins of Bordetella species. The immunogenic compositions canalso comprise at least one polypeptide of the invention in an amountsufficient to induce an immunogenic or protective response in vivo, anda pharmaceutically acceptable carrier therefor. In addition, theimmunogenic composition can comprise a neutralizing amount of at leastone polypeptide of the invention.

A preferred immunogenic composition of this invention comprises amixture of pertactins of Bordetella bronchiseptica species or fragmentsthereof, wherein the pertactins or fragments thereof comprise a mixtureof Bordetella bronchiseptica pertactin variants in which at least one ofthe Bordetella bronchiseptica pertactin variants comprises Region II ofpertactin of Bordetella bronchiseptica having 6, 7, 8, or 9 repeatingPQP amino acid sequences in Region II thereof, and at least another ofthe Bordetella bronchiseptica pertactin variants comprises Region I ofpertactin of Bordetella bronchiseptica having 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof.

In another preferred embodiment, the immunogenic composition of theinvention consists essentially of (A) a polypeptide comprising Region Iand Region II, or one polypeptide comprising Region I and onepolypeptide comprising Region II, of a pertactin of Bordetellapertussis; (B) a polypeptide comprising Region I and Region II, or onepolypeptide comprising Region I and one polypeptide comprising RegionII, of a pertactin of Bordetella parapertussis; (C) a polypeptidecomprising Region I and Region II, or one polypeptide comprising RegionI and one polypeptide comprising Region II, of a pertactin of Bordetellabronchiseptica strain 9.73 and a polypeptide comprising Region I andRegion II, or one polypeptide comprising Region I and one polypeptidecomprising Region II, of a pertactin of Bordetella bronchiseptica ofstrain SEI.

This invention also provides polynucleotides encoding the proteins andpolypeptides of the invention, as well as antibodies that recognize theproteins and polypeptides. Also provided is a DNA chip, wherein saidchip comprises at least one polynucleotide according to the invention orfragment thereof or a microarray comprising microbeads, wherein themicrobeads each bears multiple copies of a polynucleotide according toclaims 28-31 or a fragment thereof and wherein the polynucleotide orfragment thereof is different from one bead to another.

The antibodies can be monoclonal or polyclonal antibodies. Monoclonalantibodies can be used for treating Bordetella infections. Also providedare immunological complexes comprising a protein or polypeptide of theinvention and an antibody that specifically recognizes the protein orpolypeptide.

Further, this invention provides a method for detecting infection byBordetella. The method comprises providing a composition comprising abiological material suspected of being infected with Bordetella andassaying for the presence of a protein or polypeptide of the invention.The polypeptide can be assayed, for example, by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with thepolypeptide.

The method can also comprise contacting the antigen with a biologicalfluid for a time and under conditions sufficient for the antigen andantibodies in the biological fluid to form an antigen-antibody complex,and detecting the formation of the complex. The method optionally caninclude measuring the formation of the antigen-antibody complex. Inpreferred embodiments, formation of antigen-antibody complex is detectedby immunoassay based on Western blot technique, ELISA, indirectimmunofluorescence assay, or immunoprecipitation assay.

Further, this invention provides a diagnostic kit for the detection ofthe presence or absence of antibodies, which bind a protein orpolypeptide of the invention or mixtures thereof. The kit can comprisean antigen comprising the protein or polypeptide, or mixtures of theproteins and polypeptides, and means for detecting the formation ofimmune complexes between the antigen and antibodies. The means arepresent in an amount sufficient to perform the detection.

Another method of the invention for detecting the presence or absence ofBordetella comprises (1) contacting a sample suspected of containinggenetic material of Bordetella with at least one nucleotide probe, and(2) detecting hybridization between the nucleotide probe and the geneticmaterial in the sample. The nucleotide probe is complementary to apolynucleotide sequence of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail with reference to thedrawings in which:

FIG. 1 a is a map of the two regions of repeats, Region I and Region II,in the pertactin outer membrane protein of Bordetella bronchiseptica.

FIG. 1 b is an alignment of Region I of the pertactin outer membraneprotein of different strains of B. bronchiseptica.

FIG. 1 c is an alignment of Region II of the pertactin outer membraneprotein of different strains of B. bronchiseptica.

DETAILED DESCRIPTION OF THE INVENTION

It has been demonstrated previously that species-specific members of thepertactin family are outer-membrane proteins (OMPs). In B.bronchiseptica, pertactin is the product of the pm gene and isrepresented as a protein with an M_(r) of 68 kDa (P.68), in B. pertussisas a protein with an M_(r) of 69 kDa (P.69), and in B. parapertussis asa protein with an M_(r) of 70 kDa (P.70). The nucleotide sequences ofthe pertactins of these three species are included in the accompanyingSequence Listing as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3,respectively. The corresponding amino acid sequences encoded by thesenucleotide sequences are included in the sequence listing as SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

A comparison of the deduced protein sequences for the P.68, P.69. andP.70 proteins demonstrates the high degree of homology between theproteins. A comparison between the P.68 and P.70 proteins shows only 17amino acid differences, while a similar comparison between P.68 and P.69shows 80 differences, and 79 differences between P.69 and P.70. Themajority of amino acid differences between the three deduced proteinsequences occur in the number of repeat units in the two families ofrepeat sequences present in all three proteins. P.68 has three copies ofthe Gly-Gly-Xaa-Xaa-Pro repeat (i.e., GGXXP in FIG. 1 b), while P.70 hasfour and P.69 five. Similarly, P.68 has seven Pro-Gln-Pro repeats (i.e.,PQP in FIG. 1 c), P.70 has nine and P.69 has five.

It has recently been shown that the PRN produced by clinical isolates ofB. pertussis varies. Sequences of the prn gene of various clinicalisolates revealed three major types of PRN variant. It has beensuggested that epidemics in the Netherlands result from changes in thesequences of the genes encoding PRN and PT because the proteins presentin the clinical isolates currently in circulation differ in sequencefrom those observed by the vaccinal strains used in this country.

An aim of the searches, which led to the present invention, was toanalyze whether the PRN polymorphism observed in B. pertussis speciesalso occurs in B. parapertussis and B. bronchiseptica. The two repeatedregions of the prn genes of 10 B. parapertussis isolates of human originand of 40 B. bronchiseptica isolates of animal or human origin weresequenced and compared. (FIG. 1 a).

Table I contains a list of the isolates and corresponding pertactintypes used in this invention. TABLE I PRN regions I and II types/Accession Bordetella Representative Number of number,* Species isolateisolates region I, region II BB 9.73H+ I-1, II-3/3 AJ250076, AJ250077 BBLAPR I-2, II-3/8 AJ250078, AJ250079 BB  5 1-2, II-4/8 AJ250080, AJ250081BB 335 I-2, II-1/3 AJ250082, AJ250083 BB CVGEO I-2, II-5/6 AJ250084,AJ250085 BB BBCH I-2, II-6/4 AJ250086, AJ250087 BB DEL I-1, II-2/5AJ250088, AJ25089 BB CAT1 I-1, II-7/1 AJ250090, AJ250091 BB 286 I-3,II-8/1 AJ250093, AJ250092 BB SEI I-3, II-9/1 AJ250094, AJ250095 BPP 63.2 I-1, II-2/10 Identical to P24328 Species Strain PRN type Accessionnumber BPP CN2591 I-1, II-2 P24328 BB CN7531 I-2, II-4 Q03035 SpeciesStrain or isolate Allelic prn type Accession number BP Tohama prnlAJ006158 BP 18323 prn6 AJ006152 BP Hav prn2 AJ007361 BP Fr287 prn3AJ006156BB: B. bronchiseptica;BP: B. pertussis;BPP: B. parapertussis*EMBL Bank.

In carrying out this invention, DNA was extracted, amplified by PCR, andsequenced, as previously described (3). Amplified PCR products werepurified and sequenced by the ESGS company (ESGS, Cybergene group, Evry,France). Deduced amino acid sequences were analyzed with GCG software(Wisconsin Package Version 9.1, Genetics Computer Group, Madison, Wis.,USA). The deduced amino acid sequences of regions I and II were comparedand multiple alignments of the amino acid sequences were created withthe CLUSTAL W program of GCG (10), for each region (FIG. 1 b,c).

No difference was found between the sequences of regions I and II of thePRN produced by the 10 B. parapertussis isolates and the publishedsequence (15). However, three different types were found among the 40 B.bronchiseptica prn genes analyzed with differences in the number ofrepeats (1 to 3) in region I (FIG. 1 b). The largest group correspondedto sequences with three copies of the repeated sequence, identical tothe sequence previously reported (14). No correlation was found betweenthe pattern of variation and the origin of the isolate.

A higher degree of variability was observed in the second repeatedregion of the B. bronchiseptica PRN (FIG. 1 c). Nine variants wereobserved. Among these nine variants the number of repeats is from 6 to9.

No B. bronchiseptica variants presented the same pattern as the B.pertussis variants. Furthermore, no unique association between one typeof region I and one type of region II was observed. No observation wasmade in any of the three species of a pattern similar to those of the18323 strain and the CZ isolate (3), which are considered to beintermediate between B. pertussis, B. bronchiseptica, and parapertussis.These data are consistent with B. parapertussis and B. bronchisepticaprn genes being more similar to each other than to the B. pertussis prngene (1). No host specificity was observed with respect to PRN type.

It has been shown that region II plays an important role in theinduction of protective immunity (6). The lack of cross-protectionbetween PRN from B. pertussis, B. parapertussis, and B. bronchisepticaPRN is consistent with this, because the major differences between theseproteins occur in this region. No variation in this region was observedfor the PRN produced by B. pertussis isolates. These data suggest thatthirty years of vaccination may have induced variation in oneimmunodominant repeat region, but not in the region most involved in theinduction of protective immunity. Variation in B. pertussis PRN regionII may indicate a decrease in B. pertussis vaccine efficacy.

In contrast, analysis of the PRN of B. bronchiseptica showedpolymorphism in both regions. This may account for the inability of B.bronchiseptica vaccines to induce long-lasting protection. Thispolymorphism may also be linked to the ability of B. bronchiseptica toinduce chronic infections (7, 8, 22). It may provide a means for thisbacterium to escape host immune responses.

This invention, which resulted from these experiments and observations,thus involves compositions containing certain Bordetella pertactins andfragments thereof. These pertactins and pertactin fragments, as well asthe polynucleotides that encode them, are useful in immunogeniccompositions and in diagnostic applications.

In particular, this invention is the result of the discovery that thereare different species of the full length pertactin of Bordetellabronchiseptica, namely, species containing 6, 7, 8, or 9 repeating PQPamino acid sequences in Region II thereof, and species of full lengthpertactin of B. bronchiseptica containing 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof, where XX can be FD, FG, or AV.These full length pertactins and mixtures of these pertactins in anycombination of the repeating sequences are thus provided by thisinvention.

As used herein, the expression “pertactin of Bordetella bronchiseptica”means an outer membrane protein of Bordetella bronchiseptica, which is avirulence factor, and which has an apparent molecular weight of about 68kDa, and which contains the two regions of Bordetella bronchisepticapertactin known as Region I and Region II. Region I and Region II of thepertactins of different Bordetella strains are identified in brackets inSEQ ID NOS: 1 to 6. It will be understood that the pertactins ofdifferent isolates of Bordetella bronchiseptica may have amino acidsequences that differ from each other, for example, in Region I, RegionII, or both Region I and Region II, as well as in other regions.

As used herein the expression “Bordetella bronchiseptica pertactinvariants” means pertactins of Bordetella bronchiseptica, or fragments ofpertactins of Bordetella bronchiseptica containing at least Region I,Region II, or both Region I and Region II, in which the pertactins ofBordetella bronchiseptica or the fragments thereof differ from eachother in at least Region I, Region II, or both Region I and Region II,in their respective amino acid sequences. The following uniqueBordetella bronchiseptica pertactin variants have been discovered andconstitute part of this invention.

As used herein the expressions Bordetella bronchiseptica pertactinfragments”, “Bordetella parapertussis pertactin fragments”, and“Bordetella pertussis pertactin fragments” refer to polypeptides thatare portions of full length pertactin proteins and are capable ofinducing a humoral or immune response against Bordetella infections. B.bronchiseptica pertactin - region I I-1QRATIRRGDAPAGGAVPGGAVPGGAVPG---------------GFGPLLDGWYGVDVSDSTVDLAQ (SEQID NO: 7) I-2QRATIRRGDAPAGGAVPG-----GAVPG---------------GFGPLLDGWYGVDVSDSTVDLAQ (SEQID NO: 8) I-3QRATIRRGDAPAGGGVPG-----GAVPG-----GFDPGGFGPGGFGPVLDGWYGVDVSGSTVELAQ (SEQID NO: 9) prn1QRATIRRGDAPAGGAVPG-----GAVPG-----GAVPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO: 10) prn2QP.ATIRRGDAPAGGAVPG----GAVPGGFGPGGFGPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO: 11) prn3QRATIRRGDAPAGGAVPG-----GAVPG-----GFGPGGFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO: 12) prn4QRATIRRGDAPAGGAVPG-----GAVPG----------GFGPGGFGPVLDGWYGVDVSGSSVELAQ (SEQID NO: 13)**************.***     *****           ****.**********.*:*:*** B.bronchiseptica pertactin - region II II-1GAKAPPAPKPAPQPGPQPGP-----------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 14) II-2GAKAPPAPKPAPQPGPQPGPQPP--------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 15) II-3GAKAPPAPKPAPQPGPQPGPQPGPQPGP---QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 16) II-4GAKAPPAPKPAPQPGPQPGPQPGP-------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 17) II-5GAKAPPAPKPAPQPGPQPGPQPGPQP----PQPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 18) II-6GAKAPPAPKPAPQPGPQPGPQPPQPP--QPPQPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 19) II-7GAKAPPAPKPAPQPGPQP-P-----------QPPQPPQP-PQRQP--EAPAPQPPAGRELSAA (SEQ IDNO: 20) II-8GAKVPPAPKPAPQPGPQP-PQPP--------QPPQPPQPQPQPQP--EAPAPQPPAGRELSAA (SEQ IDNO: 21) II-9GAKVPPAPKPAPQPGPQP-PQPP--------QPPQPPQPQPQPQPQPEAPAPQPPAGRELSAA (SEQ IDNO: 22) prn1GAKAPPAPKPAPQPGPQP---------------PQPPQP----QP--EAPAPQPPAGRELSAA (SEQ IDNO: 23) prn6GAKAPPAPKPAPQPGPQP------------------PQP----QP--EAPAPQPPAGRELSAA (SEQ IDNO: 24)

In specific embodiments, this invention includes a polypeptidecomprising a sequence or a fragment of a sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22. The polypeptide can consistof the amino acids in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 or fragments thereof.The invention also includes polynucleotides encoding one of thesepolypeptides and a purified DNA or RNA sequence that hybridizes undermoderate or high stringency conditions to the polynucleotides or atleast to 15 nucleotides thereof.

As used herein, the expression “mixture of Bordetella bronchisepticapertactin variants” means two or more Bordetella bronchisepticapertactin variants in admixture in solid, liquid, emulsion, orsuspension form. At least two of the Bordetella bronchiseptica pertactinvariants in the mixture will, of course, differ from each other in atleast Region I, Region II, or both Region I and Region II, in theirrespective amino acid sequences.

It will be immediately apparent that this invention provides polypeptidefragments of the pertactin of B. bronchiseptica, where the fragmentscomprise 6, 7, 8, or 9 repeating PQP amino acid sequences in Region IIthereof or 1, 2, or 3 repeating GGXXP amino acid sequences in Region Ithereof. Mixtures of these polypeptide fragments in any combination ofthe repeating sequences are also within the scope of this invention.

When a polypeptide fragment of the invention comprises only Region I ofa pertactin of B. bronchiseptica, the polypeptide fragment typicallycontains at least about 46 to about 56 amino acids, which includes theRegion I repeat sequences. When the polypeptide fragment of theinvention comprises only Region II, the polypeptide fragment typicallycontains at least about 48 to about 60 amino acids, which includes theRegion II repeat sequences. When the polypeptide fragment of theinvention comprises both Region I and Region II of B. bronchiseptica,the fragment typically contains at least about 906 to about 928 aminoacids, which includes the repeat sequences of Regions I and II.

Thus, in one illustrative embodiment, this invention provides acomposition comprising a mixture of Bordetella bronchiseptica pertactinvariants, wherein each Bordetella bronchiseptica pertactin variantcomprises Region II of pertactin of Bordetella bronchiseptica, andfurther wherein each Bordetella bronchiseptica pertactin variantcomprises 6, 7, 8, or 9 repeating PQP amino acid sequences in Region IIthereof, and the Bordetella bronchiseptica pertactin variants differ inthe number of the repeating PQP amino acid sequences contained therein.The composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof. Thepolypeptide can be a full length pertactin or a fragment thereof.

In another embodiment, this invention provides a composition comprisinga mixture of Bordetella bronchiseptica pertactin variants, wherein eachBordetella bronchiseptica pertactin variant comprises Region I of apertactin of Bordetella bronchiseptica, and further wherein eachBordetella bronchiseptica pertactin variant comprises 1, 2, or 3repeating GGXXP amino acid sequences in Region I thereof, and the atleast two of the Bordetella bronchiseptica pertactin variants differ inthe number of the repeating GGXXP amino acid sequences containedtherein. This composition can also comprise pertactins of Bordetellaparapertussis, Bordetella pertussis, or mixtures thereof. The Bordetellabronchiseptica pertactin variants can be full length or a fragment.

In a further embodiment, the invention provides a composition comprisinga mixture of Bordetella bronchiseptica pertactin variants, wherein oneof the Bordetella bronchiseptica pertactin variants comprises Region IIof pertactin of Bordetella bronchiseptica having 6, 7, 8, or 9 repeatingPQP amino acid sequences in Region II thereof, and another of theBordetella bronchiseptica pertactin variants comprises Region I ofpertactin of Bordetella bronchiseptica having 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof. This composition can alsocomprise pertactins of Bordetella parapertussis, Bordetella pertussis,or mixtures thereof. The Bordetella bronchiseptica pertactin variantscan be full length or a fragment.

In a preferred embodiment, this invention provides a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, or SEQ ID NO: 9.

In another preferred embodiment, this invention provides a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

The compositions according to the invention cause a humoral immuneresponse and a cellular immune response. After infection with B.bronchiseptica, there is induction of a humoral immunity and of acellular immunity, as in the case of a B. pertussis and B. parapertussisinfection. Furthermore, after vaccination with compositions of thisinvention, there is induction of a humoral and cellular type immunitysimilar to that induced after infection or reinfection.

In one embodiment of the invention there is provided a vaccinatingcomposition comprising as active principle an immunogenic composition ofthe invention, in combination with a pharmaceutically acceptable vehicleand, where appropriate, with an adjuvant.

Like the whooping cough vaccines currently available on the market, theimmunogenic composition according to the invention may be combined withother vaccinating active principles, for example, those of the vaccineagainst diphtheria, polio, or diseases caused by Haemophilus or,generally speaking, with any immunogenic constituent, for example, aparticular inactivated pathogenic agent or toxin.

A vaccinating composition according to the invention can bespecies-specific and consequently capable of inducing protection againstB. pertussis or B. parapertussis or B. bronchiseptica. Alternatively, itcan be a mixture comprising as active principle an immunogeniccomposition against B. bronchiseptica, as defined above, and animmunogenic composition against B. parapertussis and/or B. pertussis.

As a result of recent techniques in molecular biology, a number offactors involved in the virulence of B. pertussis have beencharacterized and the regulation of their expression understood. Thesefactors may be classified in two categories, those participating in theinfectious syndrome (adhesins) and those playing a part in thetoxin-induced syndrome (toxins). The adhesins and toxins relating toBordetella can be included in the compositions of this invention.Examples of the adhesins are:

-   -   filamentous hemagglutinin or FHA, considered to play a major        part in the adhesion of the bacterium to the ciliated        epithelium;    -   the two agglutinogens or AGGs of B. pertussis, which enable        strains to be classified in serotypes; and    -   pertussis toxin or PTX, a secreted type A-B toxin which, besides        its cytopathogenic effects, participates in adhesion via its B        subunit.

Examples of the toxins for use in the invention are:

-   -   pertussis toxin or PTX, which is secreted;    -   dermonecrotic toxin or DNT, which function has not yet been well        characterized, and tracheal cytotoxin or TCT, a secreted small        glycoprotein of the muramyl peptide family, derived from the        peptidoglycan of the bacterium, which appear to act in concert        to destroy the ciliated cells of the host's respiratory        apparatus;    -   adenylate cyclase-hemolysin or Ac-Hly, a bifunctional protein        possessing adenylate cyclase activity and hemolytic activity,        which has been found to belong to the family of toxins termed        “RTX” for “repeats in toxins”.

Similarly, the factors involved in the virulence of B. parapertussis andB. bronchiseptica have been identified and can be included in thecompositions of the invention.

The published results show that the acellular vaccines tested,monovalent (PTX), bivalent (PTX, FHA), trivalent (PTX, FHA, PRN), orpentavalent (PTX, FHA, PRN, AGG2, AGG3) induce very few side effects,are all immunogenic and all have an efficacy against the disease(according to WHO definition) which is greater than or equal to 70%. Thecompositions of the invention can be included in these vaccines andother acellular vaccines. For example, the immunogenic composition canfurther comprise at least one adhesin of Bordetella selected from thegroup consisting of FHA, AGG2, AGG3, and/or at least one toxin ofBordetella selected from the group consisting of PTX, DNT, TCT, andAc-Hly.

The proteins, polypeptides, and compositions of this invention can be inpurified form. The term “purified” as used herein, means that thepertactins and fragments thereof are essentially free of associationwith other proteins or polypeptides, for example, as a purificationproduct of recombinant host cell culture or as a purified product from anon-recombinant source. The term “substantially purified” as usedherein, refers to a mixture that contains pertactins or fragmentsthereof and is essentially free of association with other proteins orpolypeptides, but for the presence of known proteins that can be removedusing a specific antibody, and which substantially purified pertactinpolypeptides can be used as antigens.

Within an aspect of the invention, the pertactin and fragments thereofcan be utilized to prepare antibodies that specifically bind topertactin polypeptides. The term “antibodies” is meant to includepolyclonal antibodies, monoclonal antibodies, fragments thereof, such asF(ab′)2 and Fab fragments, as well as any recombinantly produced bindingpartners. Antibodies are defined to be specifically binding if they bindpertactins and fragments thereof with a K_(a) of greater than or equalto about 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al., Ann. N.Y Acad Sci., 51:660 (1949).Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well known in the art.

The invention further encompasses isolated fragments andoligonucleotides derived from the nucleotide sequence of the pertactinsB. bronchiseptica, B. pertussis and B. parapertussis (SEQ ID NO: 1, SEQID NO:2, and SEQ ID NO:3) encoding 6, 7, 8, or 9 repeating PQP aminoacid sequences in Region II thereof, and/or 1, 2, or 3 repeating GGXXPamino acid sequences in Region I thereof. The invention also encompassespolypeptides encoded by these fragments and oligonucleotides. Mixturescan comprise nucleotide sequences containing repeating sequences inwhich each entity in the mixture is independently selected from thepolynucleotides of the invention.

Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native pertactinnucleic acids disclosed herein under conditions of moderate or severestringency, and which encode pertactin polypeptides. As used herein,conditions of moderate stringency, as known to those having ordinaryskill in the art, and as defined by Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring HarborLaboratory Press, (1989), include use of a prewashing solution for thenitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization conditions of 50% formamide, 6×SSC at 42° C. (or othersimilar hybridization solution, such as Stark's solution, in 50%formamide at 42° C.), and washing conditions of about 60° C., 0.5×SSC,0.1% SDS. Conditions of high stringency are defined as hybridizationconditions as above, and with washing at 68° C., 0.2×SSC, 0.1% SDS. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration can be adjusted as necessary according to factorssuch as the length of the probe.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA sequence can vary and stillencode a pertactin polypeptide having the amino acid sequence of SEQ IDNO:7 through SEQ ID NO:24. Such variant DNA sequences can result fromsilent mutations (e.g., occurring during PCR amplification), or can bethe product of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences, encodingpertactin polypeptides, selected from: (a) DNA derived from the codingregion of a native pertactin gene; (b) cDNA comprising the nucleotidesequence of SEQ ID NO:7 through SEQ ID NO:24; (c) DNA capable ofhybridization to a DNA of (a) under conditions of moderate stringencyand which encode pertactin polypeptides; and (d) DNA which is degenerateas a result of the genetic code to a DNA defined in (a), (b) or (c) andwhich encodes pertactin polypeptides. Pertactin polypeptides encoded bysuch DNA equivalent sequences are encompassed by the invention.

It will be understood that the present invention is intended toencompass the previously described proteins and polypeptides in isolatedor purified form, whether obtained using the techniques described hereinor other methods. In a preferred embodiment of this invention, thepertactin polypeptides are substantially free of human or other animaltissue and human or other animal tissue components, nucleic acids,extraneous proteins and lipids, and adventitious microorganisms, such asbacteria and viruses. It will also be understood that the inventionencompasses equivalent proteins having substantially the same biologicaland immunogenic properties. Thus, this invention is intended to coverserotypic variants of the polypeptides of the invention.

Depending on the use to be made of the pertactin polypeptides of theinvention, it may be desirable to label them. Examples of suitablelabels are radioactive labels, enzymatic labels, fluorescent labels,chemiluminescent labels, and chromophores. The methods for labeling donot differ in essence from those widely used for labelingimmunoglobulin. The need to label may be avoided by using labeledantibody to the antigen of the invention or anti-immunoglobulin to theantibodies to the antigen as an indirect marker.

Once the pertactin polypeptides of the invention have been obtained,they can be used to produce polyclonal and monoclonal antibodiesreactive therewith. Thus, a protein or polypeptide of the invention canbe used to immunize an animal host by techniques known in the art. Suchtechniques usually involve inoculation, but they may involve other modesof administration. A sufficient amount of the protein or the polypeptideis administered to create an immunogenic response in the animal host.Any host that produces antibodies to the antigen of the invention can beused. Once the animal has been immunized and sufficient time has passedfor it to begin producing antibodies to the antigen, polyclonalantibodies can be recovered. The general method comprises removing bloodfrom the animal and separating the serum from the blood. The serum,which contains antibodies to the antigen, can be used as an antiserum tothe antigen. Alternatively, the antibodies can be recovered from theserum. Affinity purification is a preferred technique for recoveringpurified polyclonal antibodies to the antigen, from the serum.

Monoclonal antibodies to the antigens of the invention can also beprepared. One method for producing monoclonal antibodies reactive withthe antigens comprises the steps of immunizing a host with the antigen;recovering antibody producing cells from the spleen of the host; fusingthe antibody producing cells with myeloma cells deficient in the enzymehypoxanthine-guanine phosphoribosyl transferase to form hybridomas;select at least one of the hybridomas by growth in a medium comprisinghypoxanthine, aminopterin, and thymidine; identifying at least one ofthe hybridomas that produces an antibody to the antigen, culturing theidentified hybridoma to produce antibody in a recoverable quantity; andrecovering the antibodies produced by the cultured hybridoma.

These polyclonal or monoclonal antibodies can be used in a variety ofapplications. Among these is the neutralization of correspondingproteins. They can also be used to detect Bordetella antigens inbiological preparations or in purifying corresponding proteins,glycoproteins, or mixtures thereof, for example when used in a affinitychromatographic columns.

The pertactin polypeptides of the invention can be used as antigens toidentify antibodies to Bordetella in materials and to determine theconcentration of the antibodies in those materials. Thus, the antigenscan be used for qualitative or quantitative determination of Bordetellain a material. Such materials, of course, include human or other animaltissue and human or other animal cells, as well as biological fluids,such as human or other animal body fluids, including human sera. Whenused as a reagent in an immunoassay for determining the presence orconcentration of the antibodies to Bordetella, the antigens of thepresent invention provide an assay that is convenient, rapid, sensitive,and specific.

More particularly, the antigens of the invention can be employed for thedetection of Bordetella by means of immunoassays that are well known foruse in detecting or quantifying humoral components in fluids. Thus,antigen-antibody interactions can be directly observed or determined bysecondary reactions, such as precipitation or agglutination. Inaddition, immunoelectrophoresis techniques can also be employed. Forexample, the classic combination of electrophoresis in agar followed byreaction with anti-serum can be utilized, as well as two-dimensionalelectrophoresis, rocket electrophoresis, and immunolabeling ofpolyacrylamide gel patterns (Western Blot or immunoblot.) Otherimmunoassays in which the antigens of the present invention can beemployed include, but are not limited to, radioimmunoassay, competitiveimmunoprecipitation assay, enzyme immunoassay, and immunofluorescenceassay. It will be understood that turbidimetric, calorimetric, andnephelometric techniques can be employed. An immunoassay based onWestern Blot technique is preferred.

Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or an antibody of theinvention to the antigen, on a carrier surface while retainingimmunoreactivity of the reagent. The reciprocal immunoreagent can beunlabeled or labeled in such a manner that immunoreactivity is alsoretained. These techniques are especially suitable for use in enzymeimmunoassays, such as enzyme linked immunosorbent assay (ELISA) andcompetitive inhibition enzyme immunoassay (CIEIA).

When either the antigen of the invention or antibody to the antigen isattached to a solid support, the support is usually a glass or plasticmaterial. Plastic materials molded in the form of plates, tubes, beads,or disks are preferred. Examples of suitable plastic materials arepolystyrene and polyvinyl chloride. If the immunoreagent does notreadily bind to the solid support, a carrier material can be interposedbetween the reagent and the support. Examples of suitable carriermaterials are proteins, such as bovine serum albumin, or chemicalreagents, such as gluteraldehyde or urea. Coating of the solid phase canbe carried out using conventional techniques.

The invention provides immunogenic pertactin polypeptides, and moreparticularly, protective polypeptides for use in the preparation ofvaccine compositions against Bordetella. These polypeptides can thus beemployed as vaccines by administering the polypeptides to a mammalsusceptible to Bordetella infection. Conventional modes ofadministration can be employed. For example, administration can becarried out by oral, respiratory, or parenteral routes. Intradermal,subcutaneous, and intramuscular routes of administration are preferredwhen the vaccine is administered parenterally.

The major purpose of the immune response in a Bordetella-infected mammalis to inactivate the Bordetella and to eliminate Bordetella infectedcells that have the potential to release infectious virus. The B-cellarm of the immune response has the major responsibility for inactivatingBordetella. The principal manner in which this is achieved is byneutralization of infectivity. Another major mechanism for destructionof the Bordetella-infected cells is provided by cytotoxic. T lymphocytes(CTL) that recognize pertactin antigens expressed in combination withclass I histocompatibility antigens at the cell surface. The CTLsrecognize pertactin polypeptides processed within cells from a pertactinprotein that is produced, for example, by the infected cell or that isinternalized by a phagocytic cell. Thus, this invention can be employedto stimulate a B-cell response to pertactin polypeptides, as well asimmunity mediated by a CTL response following infection. The CTLresponse can play an important role in mediating recovery from primaryBordetella infection and in accelerating recovery during subsequentinfections.

The ability of the pertactin polypeptides and vaccines of the inventionto induce protective levels of neutralizing antibody in a host can beenhanced by emulsification with an adjuvant, incorporating in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the pertactin polypeptides of the invention canbe administered with a conventional adjuvant, such as aluminum phosphateand aluminum hydroxide gel, in an amount sufficient to potentiatehumoral or cell-mediated immune response in the host. Similarly, thepertactin polypeptides can be bound to lipid membranes or incorporatedin lipid membranes to form liposomes. The use of nonpyrogenic lipidsfree of nucleic acids and other extraneous matter can be employed forthis purpose.

The immunization schedule will depend upon several factors, such as thesusceptibility of the host to infection and the age of the host. Asingle dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as need following the primarycourse.

The pertactin proteins, polypeptides, and vaccines of the invention canbe administered to the host in an amount sufficient to prevent orinhibit Bordetella infection or replication in vivo. In any event, theamount administered should be at least sufficient to protect the hostagainst substantial immunosuppression, even though Bordetella infectionmay not be entirely prevented. An immunogenic response can be obtainedby administering the proteins or polypeptides of the invention to thehost in an amount of, for example, about 1 to about 50 microgramsantigen per kilogram of body weight, preferably about 5 to about 10micrograms antigen per kilogram of body weight. The proteins,polypeptides, and vaccines of the invention can be administered togetherwith a physiologically acceptable carrier. For example, a diluent, suchas water or a saline solution, can be employed.

Another aspect of the invention includes administering any combinationof the nucleic acids encoding pertactin polypeptides, the proteins, andpolypeptides per se, with or without carrier molecules, to anindividual. The individual can be an animal. As used herein, the term“animal” means a mammal, and preferably, the mammal is selected from thegroup consisting of a human, a rabbit, a mouse, a dog, a cat, a bovine,a pig, and a horse. In an especially preferred embodiment, the mammal isa human.

The methods of treating include administering immunogenic compositionscomprising pertactin proteins or polypeptides, and compositionscomprising nucleic acids encoding pertactin proteins or polypeptides aswell. Those of skill in the art are cognizant of the concept,application, and effectiveness of nucleic acid vaccines (e.g., DNAvaccines) and nucleic acid vaccine technology as well as protein andpolypeptide based technologies. The nucleic acid based technology allowsthe administration of nucleic acids encoding pertactin polypeptides,naked or encapsulated, directly to tissues and cells without the needfor production of encoded proteins prior to administration. Thetechnology is based on the ability of these nucleic acids to be taken upby cells of the recipient organism and expressed to produce animmunogenic determinant to which the recipient's immune system responds.Typically, the expressed antigens are displayed on the surface of cellsthat have taken up and expressed the nucleic acids, but expression andexport of the encoded antigens into the circulatory system of therecipient individual is also within the scope of the present invention.Such nucleic acid vaccine technology includes, but is not limited to,delivery of naked DNA and RNA and delivery of expression vectorsencoding pertactin polypeptides. Although the technology is termed“vaccine”, it is equally applicable to immunogenic compositions that donot result in a protective response. Such non-protection inducingcompositions and methods are encompassed within the present invention.

Although it is within the present invention to deliver nucleic acidsencoding pertactin polypeptides and carrier molecules as naked nucleicacid, the present invention also encompasses delivery of nucleic acidsas part of larger or more complex compositions. Included among thesedelivery systems are viruses, virus-like particles, or bacteriacontaining the nucleic acid encoding pertactin polypeptides. Also,complexes of the invention's nucleic acids and carrier molecules withcell permeabilizing compounds, such as liposomes, are included withinthe scope of the invention. Other compounds, such as molecular vectors(EP 696,191, Samain et al.) and delivery systems for nucleic acidvaccines are known to the skilled artisan and exemplified in, forexample, WO 93 06223 and WO 90 11092, U.S. Pat. No. 5,580,859, and U.S.Pat. No. 5,589,466 (Vical patents), which are incorporated by referenceherein, and can be made and used without undue or excessiveexperimentation.

To further achieve the objects and in accordance with the purposes ofthe present invention, a kit capable of diagnosing a Bordetellainfection is described. This kit, in one embodiment, contains the DNAsequences of this invention, which are capable of hybridizing tobacterial RNA or analogous DNA sequences to indicate the presence of aBordetella infection. Different diagnostic techniques can be used whichinclude, but are not limited to: (1) Southern blot procedures toidentify cellular DNA which may or may not be digested with restrictionenzymes; (2) Northern blot techniques to identify RNA extracted fromcells; and (3) dot blot techniques, i.e., direct filtration of thesample through a membrane, such as nitrocellulose or nylon, withoutprevious separation on agarose gel. Suitable material for dot blottechnique could be obtained from body fluids including, but not limitedto, serum and plasma, supernatants from culture cells, or cytoplasmicextracts obtained after cell lysis and removal of membranes and nucleiof the cells by centrifugation.

Following are references of the strains used in the search concerningthe present invention:

-   -   9.73H+5, DEL, SEI: Infect Immun. (1993) 61 ”4072-4078.        Gueirard, P. and Guiso, N., filed with CNCM on May 12, 1989, No.        858.    -   CVGEO identical to strain CVHAI 286, 335: Microbiol. (1997)        143:1433-1441. Le Blay, K. et al.    -   63.2: CIP—Lab. Ident., Inst. Pasteur, Paris, France—J. Clin.        Microbiol., 1993, 31, 2745    -   TI: CIP81.32—Lab. Ident., Inst. Pasteur, Paris, France—J. Clin.        Microbiol., 1993, 31, 2746    -   Fr287: Vaccine (1999) 17:2651:2660. Boursaux-Eude, C. et al.    -   18232: ref OMS: ATCC97.97 (CIP63.1).

REFERENCES

The following references have been cited in this application. The entiredisclosure of each of these references is relied upon and incorporatedby reference herein.

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22. Woolfrey, B. F., and J. A. Moody. 1991. Human infections associatedwith Bordetella bronchiseptica. Clin. Microbiol. Rev. 4:243-255. B.bronchiseptica p.68 pertactin gene [SEQ ID NO:1] atcgatgatg cgtcgctgtaacacggcaaa taccgtgcat tgcagcggtt ctggatggcg ttcttcgtac gtttgctgcgcccattcttc cctgttccat cgcggtgcgg ccatggcggg cgtctgctct tcacccggcatccaatgaac atgtctctgt cacgcattgt cttggcggcg cccctgcgcc gcaccacactggccatggcg ctgggcgcgc tgggcgccgc gcccgccgcg tacgccgact ggaacaaccagtccatcatc aaggccggcg agcgccagca cggcatccac atcaagcaaa gcgatggcgccggcgtacgg accgccaccg gaacgaccat caaggtaagc ggtcgtcagg cccagggcgtcctgctggaa aatcccgcgg ccgagctgcg gttccagaac ggcagcgtca cgtcttcgggacagctgttc gacgaaggcg tccggcgctt tctgggcacc gtcaccgtca aggccggcaagctggtcgcc gatcacgcca cgctggccaa cgtcagcgac acccgggacg acgacggcatcgcgctctat gtggccggcg agcaggccca ggccagcatc gccgacagca ccctgcagggcgcgggcggc gtgcgggtcg agcgcggcgc caatgtcacg gtccaacgca gcaccatcgttgacgggggc ttgcatatcg gcaccctgca gccgctgcag ccggaagacc ttccgcccagccgggtggtg ctgggcgaca ccagcgtgac cgccgtgccc gccagcggcg cgcccgcggcggtgtctgta ttcggggcca atgagcttac ggttgatggc gggcacatca ccggggggcgggcagcgggg gtggcggcca tggacggggc gatcgtgcat ctg[cagcgcg cgacgatacggcggggggac gcgcctgccg gcggtgcggt tccaggcggt gctgttcccg gcggcttcggccccctcctt gacggctggt atggcgtgga tgtatcggat tccaccgtgg acctcgctcag]*tcgatcgtc gaggcgccgc agctgggcgc cgcgatccgg gcgggccgcg gcgccagggtgacggtgtcg ggcggcagct tgtccgcacc gcacggcaat gtcatcgaga ccggcggcggcgcgcgtcgc ttcccgcctc cggcctcgcc cctgtcgatc accttgcagg cgggcgcacgggcgcagggg agggcgctgc tgtaccgggt cctgccggag cccgtgaagc tgacgctggcgggcggcgcc caggggcagg gcgacatcgt cgcgacggag ctgcctccca ttccaggcgcgtcgagcggg ccgctcgacg tggcgctggc cagccaggcc * Region I cgatggacgggcgctacccg cgcggtcgac tcgctgtcca tcgacaacgc cacctgggtc atgacggacaactcgaacgt cggcgcgctg cggctggcca gcgacggcag cgtcgatttc cagcagccggccgaagctgg gcggttcaag tgcctgatgg tcgatacgct ggcgggttcg gggctgttccgcatgaatgt cttcgcggac ctggggctga gcgacaagct ggtcgtcatg cgggacgccagcggccagca caggctgttg gtccgcaaca gcggcagcga gccggccagc ggcaacaccatgctgctggt gcagacgcca cgaggcagcg cggcgacctt tacccttgcc aacaaggacggcaaggtcga tatcggtacc taccgctatc gattggccgc caacggcaat gggcagtggagcctggtg[gg cgcgaaggcg ccgccggcgc ccaagcccgc gccgcagccc ggtccccagcccggtcccca gccgccgcag ccgccgcagc cgccgcagcc gccacagagg cagccggaagcgccggcgcc gcaaccgccg gcgggcaggg agttgtccgc cgcc]**gccaac gcggcggtcaacacgggtgg ggtgggcctg gccagcacgc tctggtacgc cgaaagcaat gcgttgtccaagcgcctggg cgagttgcgc ctgaatccgg acgccggcgg cgcttggggc cgcggcttcgcgcaacgcca gcaactggac aaccgcgccg ggcggcgctt cgaccagaag gtggccggcttcgagctggg cgccgaccac gcggtggcgg tggccggcgg gcgctggcac ctgggcgggctggccggcta tacgcgcggc gaccgcggct ttaccggcga cggcggcggc cacaccgacagcgtgcatgt cgggggctat gccacctata tcgccaacag cggtttctac ctggacgcgacgctgcgcgc cagccgcctc gaaaatgact tcaaggtggc gggcagcgat gggtacgcggtcaagggcaa gtaccgcacc catggggtag gcgcctcgct cgaggcgggc cggcgcttcgcccatgccga cggctggttc ctcgagccgc aggccgagct ggcggtgttc cgggtcggcggcggttcgta ccgcgcggcc aatggcctgc gggtgcgcga cgaaggcggc agctcggtgctgggtcgcct gggcctggag gtcggcaagc gcatcgaact ggcaggcggc aggcaggtgcagccatacat caaggccagc gtgctgcagg agttcgacgg cgcgggtacg gtacgcaccaacggcatcgc gcaccgcacc gaactgcgcg gcacgcgcgc cgaactgggc ctgggcatggccgccgcgct gggccgcggc cacagcctgt atgcctcgta cgagtactcc aagggcccgaagctggccat gccgtggacc ttccacgcgg gctaccggta cagctggtaa ** Region IIagcgagaagg gtccatccCC ccgcggggga gattttcctg gaggttggcc ggtgccagtctccaggctca ggcggccagg gcgtgcgggc cgggcaggcc gtgctggtgc tggccgaacc B.bronchiseptica p.68 pertactin protein [SEQ ID NO:4] MNMSLSRIVLAAPLRRTTLA MALGALGAAP AAYADWNNQS IIKAGERQHG IHIKQSDGAG VRTATGTTIKVSGRQAQGVL LENPAAELRF QNGSVTSSGQ LFDEGVRRFL GTVTVKAGKL VADHATLANVSDTRDDDGIA LYVAGEQAQA SIADSTLQGA GGVRVERGAN VTVQRSTIVD GGLHIGTLQPLQPEDLPPSR VVLGDTSVTA VPASGAPAAV SVFGANELTV DGGHITGGRA AGVAAMDGAIVHL[QRATIRR GDAPAGGAVP GGAVPGGFGP LLDGWYGVDV SDSTVDLAQ]*S IVEAPQLGAAIRAGRGARVT VSGGSLSAPH GNVIETGGGA RRFPPPASPL SITLQAGARA QGRALLYRVLPEPVKLTLAG GAQGQGDIVA TELPPIPGAS SGPLDVALAS QARWTGATRA VDSLSIDNATWVMTDNSNVG ALRLASDGSV DFQQPAEAGR FKCLMVDTLA GSGLFRMNVF ADLGLSDKLVVMRDASGQHR LLVRNSGSEP ASGNTMLLVQ TPRGSAATFT LANKDGKVDI GTYRYRLAANGNGQWSLV[GA KAPPAPKPAP QPGPQPGPQP PQPPQPPQPP QRQPEAPAPQ PPAGRELSAA]**ANAAVNTGGV GLASTLWYAE SNALSKRLGE LRLNPDAGGA WGRGFAQRQQ LDNRAGRRFDQKVAGFELGA DHAVAVAGGR WHLGGLAGYT RGDRGFTGDG GGHTDSVHVG GYATYIANSGFYLDATLRAS RLENDFKVAG SDGYAVKGKY RTHGVGASLE AGRRFAHADG WFLEPQAELAVFRVGGGSYR AANGLRVRDE GGSSVLGRLG LEVGKRIELA GGRQVQPYIK ASVLQEFDGAGTVRTNGIAH RTELRGTRAE LGLGMAAALG RGHSLYASYE YSKGPKLANP WTFHAGYRYS W * Region I ** Region II B. pertussis p.69 gene [SEQ ID NO:2] atgaacatgtctctgtcacg cattgtcaag gcggcgcccc tgcgccgcac cacgctggcc atggcgctgggcgcgctggg cgccgccccg gcggcgcatg ccgactggaa caaccagtcc atcgtcaagaccggtgagcg ccagcatggc atccatatcc agggctccga cccgggcggc gtacggaccgccagcggaac caccatcaag gtaagcggcc gtcaggccca gggcatcctg ctagaaaatcccgcggccga gctgcagttc cggaacggca gtgtcacgtc gtcgggacag ttgtccgacgatggcatccg gcgctttctg ggcaccgtca ccgtcaaggc cggcaagctg gtcgccgatcacgccacgct ggccaacgtt ggcgacacct gggacgacga cggcatcgcg ctctatgtggccggcgaaca ggcccaggcc agcatcgccg acagcaccct gcagggcgct ggcggcgtgcagatcgagcg cggcgccaat gtcacggtcc aacgcagcgc catcgtcgac gggggcttgcatatcggcgc cctgcagtca ttgcagccgg aagaccttcc gcccagccgg gtggtgctgcgcgacaccaa cgtgaccgcc gtgcccgcca gcggcgcgcc cgcggcggtg tctgtgttgggggccagtga gcttacgctc gacggcgggc acatcaccgg cgggcgggca gcgggggtggcggccatgca aggggcggtc gtgcatctg[c agcgcgcgac gatacggcgc ggggacgcgcctgccggcgg tgcggttccc ggcggtgcgg ttcccggtgg tgcggttccc ggcggcttcggtcccggcgg cttcggtccc gtcctcgacg gctggtatgg cgtggacgta tcgggctccagcgtggagct cgcccag]*tcg atcgtcgagg cgccggagct gggcgccgca atccgggtgggccgcggcgc cagggtgacg gtgtcgggcg gcagcttgtc cgcaccgcac ggcaatgtcatcgagaccgg cggcgcgcgt cgctttgcgc ctcaagccgc gcccctgtcg atcaccttgcaggccggcgc gcatgcccag gggaaagcgc tgctgtaccg ggtcctgccg gagcccgtgaagctgacgct gaccgggggc gccgatgcgc agggcgacat cgtcgcgacg gagctgccctccattcccgg cacgtcgatc gggccgctcg acgtggcgct ggccagccag gcccgatggacgggcgctac ccgcgcggtc gactcgctgt ccatcgacaa cgccacctgg gtcatgacggacaactcgaa cgtcggtgcg ctacggctgg ccagcgacgg cagcgtcgat * Region Ittccagcagc cggccgaagc tgggcggttc aaggtcctga cggtcaatac gctggcgggttcggggctgt tccgcatgaa tgtcttcgcg gacctggggc tgagcgacaa gctggtcgtcatgcaggacg ccagcggcca gcacaggctg tgggtccgca acagcggcag cgagccggccagcgccaaca ccctgctgct ggtgcagacg ccacgaggca gcgcggcgac ctttacccttgccaacaagg acggcaaggt cgatatcggt acctatcgct atcgattggc cgccaacggcaatgggcagt ggagcctggt g[ggcgcgaag gcgccgccgg cgcccaagcc cgcgccgcagccgggtcccc agccgccgca gccgccgcag ccgcagccgg aagcgccggc gccgcaaccgccggcgggca gggagttgtc cgccgcc]**gcc aacgcggcgg tcaacacggg tggggtgggcctggccagca cgctctggta cgccgaaagc aatgcgttgt ccaagcgcct gggcgagttgcgcctgaatc cggacgccgg cggcgcctgg ggccgcggct tcgcgcaacg ccagcagctggacaaccgcg ccgggcggcg cttcgaccag aaggtggccg gcttcgagct gggcgccgaccacgcggtgg cggtggccgg cggacgctgg cacctgggcg ggctggccgg ctatacgcgcggcgaccgcg gcttcaccgg cgacggcggc ggccacaccg acagcgtgca tgtcgggggctatgccacat atatcgccga cagcggtttc tacctggacg cgacgctgcg cgccagccgcctggagaatg acttcaaggt ggcgggcagc gacgggtacg cggtcaaggg caagtaccgcacccatgggg tgggcgcctc gctcgaggcg ggccggcgct ttacccatgc cgacggctggttcctcgagc cgcaggccga gctggcggta ttccgggccg gcggcggtgc gtaccgcgcggccaacggcc tgcgggtgcg cgacgaaggc ggcagctcgg tgctgggtcg cctgggcctggaggtcggca agcgcatcga actggcaggc ggcaggcagg tgcagccata catcaaggccagcgtgctgc aggagttcga cggcgcgggt acggtacaca ccaacggcat cgcgcaccgcaccgaactgc gcggcacgcg cgccgaactg ggcctgggca tggccgccgc gctgggccgcggccacagcc tgtatgcctc gtacgagtac tccaagggcc cgaagctggc catgccgtggaccttccacg cgggctaccg gtacagctgg taa ** Region II B. pertussis p.69protein [SEQ ID NO:5] MNMSLSRIVK AAPLRRTTLA MALGALGAAP AAHADWNNQSIVKTGERQHG IHIQGSDPGG VRTASGTTIK VSGRQAQGIL LENPAAELQF RNGSVTSSGQLSDDGIRRFL GTVTVKAGKL VADHATLANV GDTWDDDGIA LYVAGEQAQA SIADSTLQGAGGVQIERGAN VTVQRSAIVD GGLHIGALQS LQPEDLPPSR VVLRDTNVTA VPASGAPAAVSVLGASELTL DGGHITGGRA AGVAAIYIQGAV VHL[QRATIRR GDAPAGGAVP GGAVPGGAVPGGFGPGGFGP VLDGWYGVDV SGSSVELAQ]*S IVEAPELGAA IRVGRGARVT VSGGSLSAPHGNVIETGGAR RFAPQAAPLS ITLQAGAHAQ GKALLYRVLP EPVKLTLTGG ADAQGDIVATELPSIPGTSI GPLDVALASQ ARWTGATRAV DSLSIDNATW VMTDNSNVGA LRLASDGSVDFQQPAEAGRF KVLTVNTLAG SGLFRMNVFA DLGLSDKLVV MQDASGQHRL WVRNSGSEPASANTLLLVQT PRGSAATFTL ANKDGKVDIG TYRYRLAANG NGQWSLV[GAK APPAPKPAPQPGPQPPQPPQ PQPEAPAPQP PAGRELSAA]**A NAAVNTGGVG LASTLWYAES NALSKRLGELRLNPDAGGAW GRGFAQRQQL DNRAGRRFDQ KVAGFELGAD HAVAVAGGRW HLGGLAGYTRGDRGFTGDGG GHTDSVHVGG YATYIADSGF YLDATLRASR LENDFKVAGS DGYAVKGKYRTHGVGASLEA GRRFTHADGW FLEPQAELAV FRAGGGAYRA ANGLRVRDEG GSSVLGRLGLEVGKRIELAG GRQVQPYIKA SVLQEFDGAG TVHTNGIAHR TELRGTRAEL GLGMAAALGRGHSLYASYEY SKGPKLAMPW T FHAGYRYSW *  Region I ** Region II B.parapertussis p.70 gene [SEQ ID NO:3] atcgatgatg cgtcgctgta acacggcaaataccgtgcat tgcagcggtt ctggatggcg ttcttcgtac gtttgctgcg cccattcttccctgttccat cgcggtgcgg gcatggcggg cgtctgCtCt tcacccggca tccaatgaacatgtctctgt cacgcattgt caaggcggcg cccctgcgcC gcaccacact ggccatggcgctgggcgcgc tgggcgccgc gcccgccgcg tacgccgact ggaacaacca gtccatcatcaaggccggcg agcgccagca cggcatccac atcaagcaaa gcgatggcgc cggcgtacggaccgccaccg gaacgaccat caaggtaagc ggtcgtcagg cccagggcgt cctgctggaaaatcccgcgg ccgagctgcg gttccagaac ggcagcgtca cgtcttcggg acagctgttcgacgaaggcg tccggcgctt tctgggcacc gtcaccgtca aggccggcaa gctggtcgccgatcacgcca cgctggccaa cgtcagcgac acccgggacg acgacggcat cgcgctctatgtggccggcg agcaggccca ggccagcatc gccgacagca ccctgcaggg cgcgggcggcgtgcgggtcg agcgcggcgc caatgtcacg gtccaacgca gcaccatcgt tgacgggggcttgcatatcg gcaccctgca gccgctgcag ccggaagacc ttccgcccag ccgggtggtgctgggcgaca ccagcgtgac cgccgtgccc gccagcggcg cgcccgcggc ggtgtttgtattcggggcca atgagcttac ggttgatggc gggcacatca ccggggggcg ggcagcgggggtggcggcca tggacggggc gatcgtgcat ctg[cagcgcg cgacgatacg gcggggggacgcgcctgccg gcggtgcggt tccaggcggt gcggttcccg gcggtgccgt tcccggcggcttcggccccc tccttgacgg ctggtatggc gtggatgtat cggactccac cgtggacctcgctcag]*tcga tcgtcgaggc gccgcagctg ggcgccgcga tccgggcggg ccgcggcgccagggtgacgg tgtcgggcgg cagcttgtcc gcaccgcacg gcaatgtcat cgagaccggcggcggtgcgc gtcgcttccc gcctccggcc tcgcccctgt cgatcacctt gcaggcgggcgcacgggcgc aggggagggc gctgctgtac cgggtcctgc cggagcccgt gaagctgacgctggcgggcg gcgcccaggg gcagggcgac atcgtcgcga cggagctgcc tcccattccaggcgcgtcga gcgggccgct cgacgtggcg ctggccagcc aggcccgatg gacgggcgctacccgcgcgg tcgactcgct gtccatcgac aacgccacct gggtcatgac ggacaactcgaacgtcggcg cgctgcggct ggccagcgac ggcagcgtcg atttccagca gccggccgaagctgggcggt tcaaggtcct gatggtcgat acgctggcgg gttcggggct gttccgcatgaatgtcttcg cggacctggg gctgagcgac aagctggtcg tcatgcggga cgccagcggccagcacaggc tgtgggtccg caacagcggc agcgagccgg ccagcggcaa caccatgctgctggtgcaga cgccacgagg cagcgcggcg * Region I acctttaccc ttgccaacaaggacggcaag gtcgatatcg gtacctaccg ctatcgattg gccgccaacg gcaatgggcagtggagcctg gtg[ggcgcga aggcgccgcc ggcgcccaag cccgcgccgc agcccggtccccagcccggt ccccagccgc cgcagccgcc gcagccgccg cagccgccgc agccgccgcagccgccacag aggcagccgg aagcgccggc gccgcaaccg ccggcgggca gggagttgtccgccgcc]**gcc aacgcggcgg tcaacacggg tggggtgggc ctggccagca cgctctggtacgccgaaagc aatgcgttgt ccaagcgcct gggcgagttg cgcctgaatc cggacgccggcggcgcttgg ggccgcggct tcgcgcaacg ccagcaactg gacaaccgcg ccgggcggcgcttcgaccag aaggtggccg gcttcgagct gggcgccgac cacgcggtgg cggtggccggcgggcgctgg cacctgggcg ggctggccgg ctatacgcgc ggcgaccgcg gctttaccggcgacggcggc ggccacaccg acagcgtgca tgtcgggggc tatgccacct atatcgccaacagcggtttc tacctggacg cgacgctgcg cgccagccgc ctcgaaaatg acttcaaggtggcgggcagc gatgggtacg cggtcaaggg caagtaccgc acccatgggg taggcgtctcgctcgaggcg ggccggcgct tcgcccatgc cgacggctgg ttcctcgagc cgcaggccgagctggcggtg ttccgggtcg gcggcggtgc gtaccgcgcg gccaatggcc tgcgggtgcgcgacgaaggc ggcagctcgg tgctgggtcg cctgggcctg gaggtcggca agcgcatcgaactggcaggc ggcaggcagg tgcagccata catcaaggcc agcgtgttgc aggagttcgacggcgcgggt acggtacgca ccaacggcat cgcgcatcgc accgaactgc gcggcacgcgcgccgaactg ggcctgggca tggccgccgc gctgggccgc ggccacagcc tgtatgcctcgtacgagtac tccaagggcc cgaagctggc catgccgtgg accttccacg cgggctaccggtacagctgg taaagcgaga agggtccatc ccccgcggag gagtttttcc tggaggttggccggtgccag tctccaggct caggcggcca gggcctgcgg gccgggcagg ccgtgctggtgctggccgaa ccattgcaca gggtgttcgg ccaagggcgg cgacttcgcc gatgaccagcaacgccgggg ggcgcacgct gcgccggcgc gcgatc ** Region I B. parapertussisp.70 protein [SEQ ID NO:6] MNMSLSRIVK AAPLRRTTLA MALGALGAAP AAYADWNNQSIIKAGERQHG IHIKQSDGAG VRTATGTTIK VSGRQAQGVL LENPAAELRF QNGSVTSSGQLFDEGVRRFL GTVTVKAGKL VADHATLANV SDTRDDDGIA LYVAGEQAQA SIADSTLQGAGGVRVERGAN VTVQRSTIVD GGLHIGTLQP LQPEDLPPSR VVLGDTSVTA VPASGAPAAVFVFGANELTV DGGHITGGRA AGVAAMDGAI VHL[QRATIRR GDAPAGGAVP GGAVPGGAVPGGFGPLLDGW YGVDVSDSTV DLAQ]*SIVEAP QLGAAIRAGR GARVTVSGGS LSAPHGNVIETGGGARRFPP PASPLSITLQ AGARAQGRAL LYRVLPEPVK LTLAGGAQGQ GDIVATELPPIPGASSGPLD VALASQARWT GATPAVDSLS IDNATWVMTD NSNVGALRLA SDGSVDFQQPAEAGRFKVLM VDTLAGSGLF RMNVFADLGL SDKLVVMRDA SGQHRLWVRN SGSEPASGNTMLLVQTPRGS AATFTLANKD GKVDIGTYRY RLAANGNGQW SLV[GAKAPPA PKPAPQPGPQPGPQPPQPPQ PPQPPQPPQP PQRQPEAPAP QPPAGRELSA A]**ANAAVNTGG VGLASTLWYAESNALSKRLG ELRLNPDAGG AWGRGFAQRQ QLDNPAGRRF DQKVAGFELG ADHAVAVAGGRWRLGGLAGY TRGDRGFTGD GGGHTDSVHV GGYATYIANS GFYLDATLRA SRLENDFKVAGSDGYAVKGK YRTHGVGVSL EAGRRFAHAD GWFLEPQAEL AVFRVGGGAY RAANGLRVRDEGGSSVLGRL GLEVGKRIEL AGGRQVQPYI KASVLQEFDG AGTVRTNGIA HRTELRGTRAELGLGMAAAL GRGHSLYASY EYSKGPKLAN PWTFHAGYRY SW *  Region I ** Region II

1-55. (canceled)
 56. A polypeptide comprising a sequence or a fragmentof the sequence of: SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, wherein thesequence or fragment of the sequence includes the Region I or Region IIrepeat sequences.
 57. A polypeptide consisting of the amino acids in SEQID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, or SEQ ID NO:
 22. 58. A diagnostic kit for the detection ofthe presence or absence of antibodies, which bind a polypeptide of claim56 or mixtures thereof, wherein the kit comprises an antigen comprisinga polypeptide of claim 56 or mixtures of said polypeptides, and meansfor detecting the formation of immune complex between the antigen andantibodies, wherein the means are present in an amount sufficient toperform said detection.
 59. An immunogenic composition comprising atleast one polypeptide of claim 56 in an amount sufficient to induce animmunogenic or protective response in vivo, and a pharmaceuticallyacceptable carrier therefor.